The main storage lipids in plants are triacylglycerols (TAGs) which are present in most plant organs: developing seeds, flower petals, anthers, pollen grains, and fruits (Stymne and Stobart, 1987; Oo and Chew 1992; Xue et al., 1997; Murphy and Vance, 1999). TAGs are thought to be not only the major energy source for seed germination but also essential for pollen development and sexual reproduction in many plants (Slocombe et al., 1994; Wolters-Arts et al., 1998; Zheng et al., 2003). TAG bioassembly is catalyzed by the membrane-bound enzymes of the Kennedy pathway that operate in the endoplasmic reticulum (Stymne and Stobart, 1987). The process begins with sn-glycerol-3-phosphate (G3P) undergoing two acylations catalyzed by the acyltransferases glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) and lysophosphatidic acid acyltransferase (LPAAT; EC 2.3.1.51) The final acylation of sn-1,2-DAG by diacylglycerol acyltransferase (DGAT; EC 3.2.1.20) to give TAG, occurs after removal of the phosphate group from the sn-3 position of the glycerol backbone by phosphatidate phosphatase (PAPase; EC 3.1.3.4). It has been suggested that DGAT may be one of the rate-limiting steps in plant storage lipid accumulation (Ichihara et al., 1988; Perry and Harwood, 1993a & b; Perry et al., 1999; Jako et al., 2001; Weselake R J, 2005; Lung and Weselake, 2006), and thus a potential target in the genetic modification of plant lipid biosynthesis in oilseeds for economic benefit.
In the traditional Kennedy pathway DGAT is the only enzyme that is exclusively committed to TAG biosynthesis using acyl-CoA as its acyl donor. The first DGAT gene was cloned from mouse and is a member of the DGAT1 family, which has high sequence similarity with sterol:acyl-CoA acyltransferase (Cases et al., 1998). A second family of DGAT genes (DGAT2) was first identified in the oleaginous fungus Morteriella ramanniana, which has no sequence similarity with DGAT1 (Lardizabal et al., 2001). A novel class of acyl-CoA-dependent acyltransferases, wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT) was recently identified and purified from the bacterium Acinetobacter sp. strain ADP1, which can utilize both fatty alcohols and diacylglycerols as acyl acceptors to synthesize wax esters and TAGs, respectively (Kalscheuer and Steinbuchel, 2004; Stoveken et al., 2005). Other proposed additions to the traditional scheme of the Kennedy pathway include demonstrations that in developing castor and safflower seeds, TAG can also be generated from two molecules of DAG via a DAG:DAG transacylase (with MAG as a co-product) and that the reverse reaction participates in remodeling of TAGs (Lehner and Kuksis, 1996; Mancha and Stymne, 1997; Stobart et al, 1997). In some species, it is apparent that TAG can also be formed by an acyl-CoA-independent enzyme, phosphatidylcholine:diacylglycerol acyltransferase (PDAT), in which the transfer of an acyl group from the sn-2 position of PC to the sn-3 position of DAG yields TAG and sn-1 lyso-PC (Dahlqvist et al, 2000; Banas et al., 2000). The two closest homologs to the yeast PDAT gene have been identified in Arabidopsis (Stahl et al, 2004). These findings suggest that these other TAG synthesizing enzymes may regulate the TAG biosynthesis at different stages of seed development or in different cellular compartments. It is not yet clear to what extent these enzymes may play a role in conventional TAG assembly in oilseeds. For example, Mhaske et al (2005) isolated and characterized a knockout mutant of Arabidopsis thaliana L. which has a T-DNA insertion in PDAT locus At5g13640 (PDAT, EC 2.3.1.158). Lipid analyses were conducted on these plants to assess the contribution of the PDAT gene to lipid composition; surprisingly, the fatty acid content and composition in seeds did not show significant changes in the mutant. This is a contrary situation to yeast where PDAT is a major contributor to triacylglycerol (TAG) accumulation in exponential growth phase. The results were interpreted to indicate that PDAT activity as encoded by At5g13640 is not a major determining factor for TAG synthesis in Arabidopsis seeds. Nonetheless, these other TAG synthesizing enzymes may regulate TAG biosynthesis at different stages of seed development or in different cellular compartments (Marianne et al., 2002).
We previously characterized an EMS-induced mutant of Arabidopsis, designated AS11, which displayed a decrease in stored TAG and an altered fatty acid composition (Katavic et al., 1995). Since the first identification of the DGAT1 gene from Arabidopsis (Zou et al., 1999; Hobbs et al., 1999; Routaboul et al., 1999), homologous DGAT1 genes from several other plants have been cloned (Bouvier-Nave et al, 2000; Nykiforuk et al., 2002; He et al., 2004; Milcamps et al., 2005; Wang et al., 2006; Saha et al., 2006; Shockey et al., 2006). Studies on these genes showed that the DGAT1 plays a dominating role in determining oil accumulation and fatty acid composition of seed oils. Thus, there was implied utility in manipulating the expression of this gene for improving oil content and perhaps, altering fatty acid composition. To this end, we demonstrated that expression of the Arabidopsis DGAT1 cDNA in a seed specific manner in the AS11 mutant restored wild type levels of TAG and VLCFA content. The acyl distribution i.e. the sn-3 composition of the TAGs was also restored to WT. Furthermore, over-expression of the Arabidopsis DGAT1 in wild type plants led to an increase in seed oil content and seed weight (Jako et al., 2001).
Oilseeds produce a variety of chemically unusual fatty acids that are currently used as industrial feedstocks. Erucic acid (22:1Δ13) is one such fatty acid, and high erucic acid rapeseed (HEAR) is grown as an industrial feedstock crop on the Canadian prairies. The industrial applications of high erucic acid seed oils and their derivatives include lubricants, slip-promoting agents (in the manufacture of plastic films), nylon 1313, plasticizers, coating agents, photographic developers etc. (Taylor et al., 2001) The current market for high erucate oils exceeds $120M U.S./annum. Consistent with the market trends predicted by Sonntag (1995), since 1990, worldwide erucic acid demand has almost doubled and is predicted to reach 80 million pounds by the year 2010. Similarly, demand for the derivative behenic acid is predicted to triple to about 102 M pounds by 2010. Similarly, demand for the derivative, behenic acid, is predicted to triple to about 102 M pds by 2010. In recent years, production has increased to meet market needs, and high erucic acreage in western Canada is currently at a record high (D. Males, Saskatchewan Wheat Pool, personal communication). A Brassica cultivar containing erucic acid levels approaching 80% would significantly reduce the cost of producing erucic acid and its derivatives, and could meet the forecast demand for erucic and behenic acids as renewable, environmentally friendly industrial feedstocks (Leonard, 1994; Taylor et al., 2001; Mietkiewska et al., 2004). For this reason, improving the erucic acid content of HEAR Brassicaceae is of interest in a biotechnology context. Erucic acid is synthesized by successive 2-carbon extensions of oleic acid donated from malonyl-CoA by the action of an elongase complex (Katavic et al., 2001).
The only plant known to accumulate trierucin in its seed oil is garden nasturtium (Tropaeolum majus). Although the total oil content of the seed is only 8-15%, erucic acid constitutes 70-75% of the total fatty acid composition and most of this is in the form of trierucin (Pollard and Stumpf, 1980, Taylor et al., 1992).
There is a need in the art to isolate a gene from Tropaeolum majus that encodes the DGAT1 protein.